PROJECT SUMMARY/ABSTRACT The goals of this proposal are: (1) to elucidate specific mechanisms by which hematopoietic (blood-forming) stem cells (HSCs) and factors within their microenvironment (i.e., niche) lead to age-associated changes in blood formation; and (2) to identify related HSC and niche mechanisms that enhance HSC engraftment after transplantation. More specifically, we aim to achieve these goals, respectively: (1) by manipulating Lnk (Sh2b3) expression in HSCs to identify intrinsic regulators of HSC aging and the myeloid-bypass differentiation pathway (see below); and (2) by identifying ?supporter? cells in the niche, and their corresponding molecular mechanisms, that enable HSC engraftment. This project will provide a mechanistic basis to devise strategies to treat age- associated hematologic disorders and improve methods of HSC transplantation (HSCT), thereby addressing two important medical needs. HSCT is a curative therapy for a number of hematological diseases (including immunodeficiencies, autoimmunities, and leukemias) but is still risky, in part due to the necessary pre-HSCT bone marrow conditioning, usually irradiation or chemotherapy. Such conditioning is necessary to make ?space? or clear niches in the bone marrow for donor HSCs to engraft, but also causes significant morbidity. With the overall goal of improving our understanding of HSC-niche interactions, this research program: (1) will identify novel strategies to sustain HSC function during aging, potentially preventing or treating age-associated hematologic disorders; and (2) to improve HSC transplantation, potentially by reducing the need for toxic pre- conditioning. Aim 1 is based on findings that: (i) Lnk-deficient HSCs do not exhibit the typical age-associated myeloid bias of wild-type HSCs; (ii) within the immunophenotypically-defined HSC compartment are functionally distinct self-renewing myeloid-restricted progenitor cells (MyRPs) that are directly generated from HSCs through a myeloid-bypass pathway; and (iii) MyRPs increase in frequency with age. Through analysis of Lnk-deficient HSCs, which are defective in MyRP generation, we will identify mechanisms regulating myeloid-bypass differentiation. From transcriptomics analysis, we will identify new putative regulators of MyRP production and HSC aging, and confirm these through functional assays. Aim 2 is based on the finding that Lnk-deficient HSCs have greater engraftment potential than wild-type HSCs in HSCT, even in the absence of preconditioning. We will identify non-HSC bone marrow cell types that enhance HSC engraftment. We elucidate cellular mechanisms that contribute to this ?supporter cell? function using 2D and 3D microscopy to trace bone marrow colonization following transplantation. In summary, this research proposal will use Lnk-deficiency as a system to both understand fundamental mechanisms of HSC aging and identify strategies to improve HSC transplantation engraftment rates. This research project will build on the long-term objectives of improving clinical transplantation and healthy aging through better understanding of stem cell function and the stem cell niche.